TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

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Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽

10.1182/blood-2018-99-115066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Jasna Marjanovic ◽

Brad Rumancik ◽

Luke Weber ◽

Felix Wangmang ◽

Dane Fickes ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Thrombus Formation ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

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Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo

Blood ◽

10.1182/blood-2005-09-3687 ◽

2006 ◽

Vol 107 (10) ◽

pp. 3902-3906 ◽

Cited By ~ 158

Author(s):

Christophe Dubois ◽

Laurence Panicot-Dubois ◽

Glenn Merrill-Skoloff ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Severe Injury ◽

Wild Type ◽

Vessel Occlusion ◽

Platelet Surface ◽

Glycoprotein Vi ◽

Laser Injury ◽

Platelet Accumulation

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRγ-null mice (FcRγ–/–) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl3 injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl3 injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRγ–/– compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRγ–/–. These data indicate a significant role for GPVI in FeCl3-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl3-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRγ–/– was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl3-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

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Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Blood ◽

10.1182/blood.v108.11.1510.1510 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1510-1510

Author(s):

Christophe Dubois ◽

Laurence Panicot-Dubois ◽

Justin F. Gainor ◽

Barbara C. Furie ◽

Bruce Furie

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Specific Inhibitor ◽

Calcium Mobilization ◽

Wild Type ◽

Platelet Surface ◽

Platelet Thrombus ◽

Platelet Accumulation ◽

Kinetics Of

Abstract Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood.2006;107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of α2β1 but not αIIbβ3 integrin

Thrombosis and Haemostasis ◽

10.1160/th04-12-0783 ◽

2005 ◽

Vol 94 (07) ◽

pp. 107-114 ◽

Cited By ~ 21

Author(s):

Christelle Lecut ◽

Martine Jandrot-Perrus ◽

Marion A. H. Feijge ◽

Judith M. E. M. Cosemans ◽

Johan W. M. Heemskerk

Keyword(s):

Thrombin Generation ◽

Type I Collagen ◽

Platelet Rich Plasma ◽

Procoagulant Activity ◽

Type I ◽

Platelet Surface ◽

Glycoprotein Vi ◽

Collagen Receptors ◽

Fibrillar Collagens

SummaryThe role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin α2β1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the α2β1 activation state. Blockade of α2β1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of αIIbβ3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) α2β1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) αIIbβ3 is not directly involved in the collagen effect.

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Nucleotide Polymorphisms in the α2 Gene Define Multiple Alleles That Are Associated With Differences in Platelet α2β1 Density

Blood ◽

10.1182/blood.v92.7.2382.2382_2382_2388 ◽

1998 ◽

Vol 92 (7) ◽

pp. 2382-2388 ◽

Cited By ~ 9

Author(s):

Marcie Kritzik ◽

Brian Savage ◽

Diane J. Nugent ◽

Sentot Santoso ◽

Zaverio M. Ruggeri ◽

...

Keyword(s):

Whole Blood ◽

Type I Collagen ◽

Type I ◽

Nucleotide Polymorphisms ◽

Coding Region ◽

Platelet Surface ◽

Multiple Alleles ◽

Increased Risk ◽

Allelic Differences

Three allelic differences in the α2 gene are associated with expression levels of the α2β1 integrin on the platelet surface. We have previously defined two linked silent polymorphisms in the α2 gene coding region at nucleotides 807 (C or T) and 873 (G or A). We have now identified one rarer nucleotide polymorphism in the coding region at nucleotide 837 (T or C) and four additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we have determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism. Thus, three α2 gene alleles, defined by eight nucleotide polymorphisms, have now been discovered. Allele 1 (807T/837T/873A/Brb) is associated with increased levels of α2β1; allele 2 (807C/837T/873G/Brb) and allele 3 (807C/837C/873G/Bra) are each associated with lower levels of α2β1. Finally, we also show here that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (1,500/s) is proportional to the density of α2β1 receptors on the platelet surface. Thus, the density of platelet α2β1 could have an important impact on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.

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CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Blood ◽

10.1182/blood.v104.11.326.326 ◽

2004 ◽

Vol 104 (11) ◽

pp. 326-326

Author(s):

Wolfgang Bergmeier ◽

Jill R. Crittenden ◽

Crystal L. Piffath ◽

Denisa D. Wagner ◽

David E. Housman ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Knockout Mice ◽

Thrombus Formation ◽

Es Cells ◽

Embryonic Stem ◽

Premature Stop Codon ◽

Small Gtpase ◽

Integrin Αiibβ3

Abstract Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.

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Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽

10.1182/blood.v112.11.111.111 ◽

2008 ◽

Vol 112 (11) ◽

pp. 111-111 ◽

Cited By ~ 1

Author(s):

Meghna Ulhas Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas Pandurang Naik

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Adhesion Molecule ◽

Resting State ◽

Human Platelets ◽

Artery Occlusion ◽

In Vivo Model ◽

Platelet Surface ◽

Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

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Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Blood ◽

10.1182/blood-2011-07-364992 ◽

2012 ◽

Vol 119 (3) ◽

pp. 847-856 ◽

Cited By ~ 47

Author(s):

Alessandra Consonni ◽

Lina Cipolla ◽

Gianni Guidetti ◽

Ilaria Canobbio ◽

Elisa Ciraolo ◽

...

Keyword(s):

Knockout Mice ◽

Type I Collagen ◽

Thrombus Formation ◽

Human Platelets ◽

Time Dependent ◽

Type I ◽

Integrin Αiibβ3 ◽

Akt Phosphorylation ◽

Inside Out ◽

Stimulation Of

Abstract Integrin α2β1–mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3KβKD), but occurred normally in PI3KγKD platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)–knockout mice, and we found that intracellular Ca2+ linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca2+. Whereas activation of Pyk2 occurred normally in PI3KβKD platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1–mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.

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Dual role of collagen in factor XII–dependent thrombus formation

Blood ◽

10.1182/blood-2008-07-171066 ◽

2009 ◽

Vol 114 (4) ◽

pp. 881-890 ◽

Cited By ~ 134

Author(s):

Paola E. J. van der Meijden ◽

Imke C. A. Munnix ◽

Jocelyn M. Auger ◽

José W. P. Govers-Riemslag ◽

Judith M. E. M. Cosemans ◽

...

Keyword(s):

Thrombin Generation ◽

Type I Collagen ◽

Factor Viia ◽

Thrombus Formation ◽

Dual Role ◽

Type I ◽

Factor Xii ◽

Glycoprotein Vi

Abstract In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

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